Pathological Features of Echovirus-11-Associated Brain Damage in Mice Based on RNA-Seq Analysis

Figures

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  Figure 1. Body weights, survival rates of mice, clinical score percentage, and symptoms in the experiments. Two-day-old IFNAR−/− mice (n = 16 per group) were inoculated intracranially with 40 µL of E11 suspension (virus titre, 108 TCID50/mL, diluted 10-fold to 107 TCID50/mL). Control animals were administered culture medium instead of virus. Body weight (A), survival rates (B), clinical score percentage (C), and clinical symptoms (D) were monitored and recorded daily after inoculation with E11 until 6 dpi. Data represent the mean results of 16 mice ± standard error of the mean; * p < 0.05; ** p < 0.01; NC, negative control.

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  Figure 2. Changes in E11 viral load in different tissues at 2, 4, and 5 dpi. Two-day-old neonatal IFNAR−/− mice were challenged with 40 µL of E11 strain HuB2019-6(virus titre, 108 TCID50/mL, diluted 10-fold to 107 TCID50/mL) and the viral loads in the brain, hind limb muscle, heart, lung, intestine, and spleen tissues were determined by TCID50. Data represent the mean results of three mice ± standard error of the mean with five biological replicates.

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  Figure 3. Histopathological examination. After the two-day-old neonatal IFNAR−/− mice were challenged intracranially with 40 µL of E11 suspension (virus titre, 108 TCID50/mL, diluted 10-fold to 107 TCID50/mL), tissue samples from the brain (A 1 to 6), hind limb muscle (B 1 to 6), heart (C 1 to 6), lung (D 1 to 6), and intestine (E 1 to 6) were subjected to pathological analysis. No obvious histological changes (1, 2) were observed in each tissue of the negative control group. Mice with grade 4 or 5 clinical symptoms infected with E11(HuB2019-16) exhibited cerebral oedema (A); massive damage was observed in the limb muscles, characterised by the foci of myositis and myonecrosis associated with neutrophilic infiltration (B); alveolar shrinkage, pulmonary fibrosis, and viral pneumonia in the lung tissue (C); severe myocardial tears and capillary leakage in the cardiac tissue (D); and vacuolar degeneration of small intestinal villi accompanied by massive inflammatory cell infiltrates (E); viral hepatitis is characterized by hepatocellular edema with lymphocytic infiltration (F); nephritis is characterized by glomerulocytosis (G). Observations of singular columns (1, 3, 5) were made at a…

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  Figure 4. Visualisation of gene expression changes and their biological pathways. Transcriptome analysis of the brain and skeletal muscle tissues from 2-day-old mice infected with 107 TCID50 via intracranial injection at 4 dpi. The detailed number of upregulated and downregulated differentially expressed genes (DEGs) in each group (A). Venn diagram displaying the number of brain-specific, muscle-specific, and brain/muscle-common DEGs after E11 inoculation (B). GO functional analysis and KEGG pathways of differentially expressed genes in the brain and skeletal muscle (C,D). Visualisation of DEGs involved in antiviral and other related immune response pathways (E,F).

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  Figure 5. Virus replication in mouse brain tissues and neural cells. (A) Brain tissue samples of 2-day-old mice challenged with 107 TCID50 via intracranial injection, sacrificed at 4 dpi, stained with dsRNA against E11 (red), and counterstained with DAPI (blue) for detection of nuclei. Scale bar = 50 μm. (B) U251 and SK-N-SH cells were infected with E11 at an MOI of 10 as described above. Cells were visualised at 24 hpi under bright-field microscopy at 5/10× magnification. (C) Infection and replication of E11 virus in U251 and RD cells infected with MOI = 10/0.005, respectively. Infection medium was removed at 2 hpi to reduce background. Cell culture lysates were collected at four time points (12 hpi, 24 hpi, 48 hpi, and 72 hpi) after infection, and viral titres were measured using endpoint dilutions for growth in RD cells. Error bars represent standard error of the mean (SEM) from three biological replicates. (D) U251 cells were infected with E11 at an MOI of 10. Cells were fixed at 24 hpi and stained with FITC (green) against E11 and counterstained with DAPI (blue) for the detection of nuclei.